In vitro antiproliferative activity of 11-aminoalkylamino-substituted 5H-indolo[2,3-b]quinolines; Improving activity of neocryptolepines by installation of ester substituent

Article abstract of DOI:10.1007/s00044-012-0443-x

The research article describes the effect of an ester group on the in vitro antiproliferative activity in SAR studies of 5-methyl-5H-indolo[2,3-b]quinoline (neocryptolepine) derivatives. The C-2 and/or C-9 ester-substituted neocryptolepines were synthesized starting from indole-3-carboxylates and N-methylanilines, which were bearing an ester group. To these ester-substituted neocryptolepines, various aminoalkylamino substituents were further attached at the C-11, and an in vitro antiproliferative assay was performed by varying the substituents at the C-11 and the position of the ester group in the A and/or D ring of neocryptolepines. Results indicated that the antiproliferative activities of the agents could be improved by introducing an ester substituent at the C-9 position. Among them, the methyl 11-(3-aminopropylamino)-5-methyl-5H- indolo[2,3-b]quinoline-9-carboxylate (8b) was the most potent agent with an IC₅₀ value of 0.044 μM against the human leukemia MV4-11 cell line. The selective cytotoxicity of the agents between the cancer cell lines and normal cell lines were also described. The antiproliferative potency of dimethyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-2,9- dicarboxylate (9a) against the human colon cancer cell line HCT116 is 28 times higher than against the normal mice fibroblast cell line BALB/3T3. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0443-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4492–4504
DOI 10.1007/s00044-012-0443-x
ORIGINAL RESEARCH
In vitro antiproliferative activity of 11-aminoalkylamino-
substituted 5H-indolo[2,3-b]quinolines; improving activity
of neocryptolepines by installation of ester substituent
´
Wen-Jie Lu Marta Switalska Li Wang Mizuki Yonezawa
•
•
•
•
•
Ibrahim El-Tantawy El-Sayed Joanna Wietrzyk Tsutomu Inokuchi
•
Received: 14 September 2012 / Accepted: 18 December 2012 / Published online: 12 January 2013
Ó Springer Science+Business Media New York 2013
Abstract The research article describes the effect of an
ester group on the in vitro antiproliferative activity in SAR
studies of 5-methyl-5H-indolo[2,3-b]quinoline (neocry-
ptolepine) derivatives. The C-2 and/or C-9 ester-substituted
neocryptolepines were synthesized starting from indole-3-
carboxylates and N-methylanilines, which were bearing an
ester group. To these ester-substituted neocryptolepines,
various aminoalkylamino substituents were further
attached at the C-11, and an in vitro antiproliferative assay
was performed by varying the substituents at the C-11 and
the position of the ester group in the A and/or D ring of
neocryptolepines. Results indicated that the antiprolifera-
tive activities of the agents could be improved by intro-
ducing an ester substituent at the C-9 position. Among
them, the methyl 11-(3-aminopropylamino)-5-methyl-
5H-indolo[2,3-b]quinoline-9-carboxylate (8b) was the
most potent agent with an IC50 value of 0.044 lM against
the human leukemia MV4-11 cell line. The selective
cytotoxicity of the agents between the cancer cell lines and
normal cell lines were also described. The antiproliferative
potency of dimethyl 11-(3-aminopropylamino)-5-methyl-
5H-indolo[2,3-b]quinoline-2,9-dicarboxylate (9a) against
the human colon cancer cell line HCT116 is 28 times
higher than against the normal mice fibroblast cell line
BALB/3T3.
Keywords 5H-indolo[2,3-b]quinoline Á
Anti-proliferative agent Á
C9-ester substituted necryptolepine Á MV4-11 Á HCT116
Introduction
Indoloquinolines isolated from the root of Cryptolepis
sanguinolenta, the climbing shrub growing in some Afri-
can countries, are attracting the constant attention of
pharmacologists, since an aqueous macerate or decoction
of this root is used in traditional medicine for the treatment
of infectious diseases like malaria and various disorders of
the body (Alexandra et al., 2000; Cimanga et al., 1996a, b).
Among the various isolated products with indoloquinoline
structures, cryptolepine i (5-methylindolo[3,2-b]quinoline),
and the minor alkaloid neocryptolepine ii (5-methylindo-
lo[2,3-b]quinoline) (Fig. 1) have been intensively studied,
which showed broad spectrum of biological activities
including antibacterial (Cimanga et al., 1996a, b, 1997,
1998), antifungal (Cimanga et al., 1996a, b, 1997, 1998),
and antimalarial (Philippe et al., 1996; Kirby et al., 1995;
Wright et al., 1996).
W.-J. Lu Á L. Wang Á M. Yonezawa Á I. E.-T. El-Sayed Á
T. Inokuchi (&)
Division of Chemistry and Biotechnology, Graduate School of
Natural Science and Technology, Okayama University, 3-1-1
Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
e-mail: inokuchi@cc.okayama-u.ac.jp
Since cryptolepine i and neocryptolepine ii possess
linearly arranged tetracyclic planar structures, they behave
as a DNA intercalating agent and inhibit topoisomerase II
thus revealing a high level of cytotoxicity (Guittat et al.,
2003; Jonckers et al., 2002; Bailly et al., 2000). Conse-
quently, SAR studies on these structures as a lead to search
for more active drug candidates have been extensively
´
M. Switalska Á J. Wietrzyk (&)
Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, 12, R. Weigl Street, 53-114 Wroclaw,
Poland
I. E.-T. El-Sayed
Chemistry Department, Faculty of Science, El Menoufeia
University, Shebin El Koom, Egypt
123

Med Chem Res (2013) 22:4492–4504
4493
undertaken (Kumar et al., 2008; Lavrado et al., 2010;
Parvatkar et al., 2011).
agents such taxol (Ojima et al., 1997), camptothecin
(Yang et al., 2002), bryostatin (Wender and Hinkle,
2000), and 5-fluorouracil (Xiong et al., 2009). Such an
improvement in the anticancer activity is believed to be
due to increasing both the lipophilicity and bioavailability
of the drug by introducing the ester group into the core
structure. Encouraged by these significant information, we
examined the introduction of an ester group to the core
and checked its influence on the antiproliferative activity.
In this study, we report the further investigation of the
anticancer potential of the neocryptolepine core. An ester
group was introduced at the C-2 position of the A-ring
and/or the C-9 position of the D-ring to establish or
extend the structure-activity relationship (SAR) study for
these regions.
Our previous research on 5-Me-indolo[2,3-b]quinolines
revealed that the 11-amino group is important for their
activity, especially the 3-aminopropylamino group, which
could increase the activity against MV4-11 about 67 times
compared to its 11-chloro precursor (Wang et al., 2012).
The antiproliferaive test indicated that the 5-methylated
derivatives are usually more cytotoxic than their respective
6-methylated congeners. A synergistic effect of the sub-
stituents at the C-2 position was also observed. For
example, the C-2-Br derivative was 5 times more active
compared to the non-halogenated derivative on 11-(3-am-
inopropylamino)neocryptolepine, and the 2-chloro-11-(4-
methyl-1,4-diazepane-1-yl)neocryptolepine was 65 times
more active than its corresponding agent with no sub-
stituent at C-2.
On the other hand, a recent publication describing
effect of substituents on the anticancer activity indicated
that the introduction of an ester group to the core struc-
tures affects the activity of other plant-derived anticancer
Synthesis and biological evaluation
The preparations of the ester substituted 11-chloro-
5-methyl-5H-indolo[2,3-b]quinolines 6 were carried out
according to the method we previously described (Wang
et al., 2012). Since the dimethyl 1H-indole-3,5-dicarbox-
ylate (2b), a key starting compound, was not commercially
available, it was synthesized by installation of an ester
group at the C-3 position to the methyl 1H-indole-5-car-
boxylate 1 using the reaction with trichloroacetyl chloride
in the presence of pyridine, followed by successive
alkaline treatment with KOH at reflux in MeOH and MeI
N
D
A
C
B
N
N
N
CH3
CH3
i Cryptolepine
ii Neocryptolepine
Fig. 1 Structures of cryptolepine and neocryptolepine
Scheme 1 Preparation of ester
substituted 11-amino-
neocryptolepines 7–9. Reagents
and conditions: (i) a. Pyridine,
trichloroacetyl chloride, THF.
b. KOH, MeOH, reflux.
(ii) a. N-chlorosuccinimide,
1,4-dimethylpiperazine.
b. trichloroacetic acid. (iii)
diphenyl ether, reflux. (iv)
POCl3, toluene, reflux.
(v) appropriate amines
123

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Med Chem Res (2013) 22:4492–4504
Table 1 Compounds 7–8 as anti-proliferative agents against human leukemia cell line MV4-11
R3
R2
R2
R1
N
N
N
N
Me
Me
10
7-8
IC50 (µM)
IC50 (µM)
Compound
7–8
Yield
%
R1
R2
R3
Compound 10
R2
MV4-11
MV4-11
0.006 0.002
0.086 0.020
Doxorubicin HCl
7
–CO2Me
H
HN
NH2
H
93
89
-CO2Me
0.094 0.030
10aa
0.170 0.010
8a
HN
HN
OH
HN
HN
OH
H
H
NH2
-CO2Me
-CO2Me
92
90
0.044 0.011
0.797 0.227
10ba
10ca
NH2 0.066 0.023
0.164 0.047
8b
8c
O
N
O
N
10da
7.171 0.987
CH3
N
CH3
N
H
-CO2Me
91
0.127 0.035
8d
N
N
10ea
10fa
0.100 0.035
1.312 0.262
NH2
NH2
H
H
H
-CO2Me
-CO2Me
-CO2Me
95
86
87
0.069 0.019
0.811 0.250
0.051 0.023
8e
8f
HN
HN
Me
Cl
N
HN
Me
Me
8g
HN
N
Me
a
Wang et al. (2012)
in DMF (Linton and Kozlowski, 2008). The obtained
dimethyl 1H-indole-3,5-dicarboxylate (2b) was combined
with N-methylaniline (3b) via chlorination with NCS in the
presence of 1,4-dimethylpiperazine, giving 2-(N-methy-
lanilino)indole-3,5-dicarboxylate (4b) in 73 % yield.
Heating of 4b at 250 °C in diphenyl ether induced intra-
molecular acylation at the C-2 of aniline, forming the tet-
racyclic indolo[2,3-b]quinolinone 5. The reaction of 5 with
POCl3 afforded the 11-chloro-indolo[2,3-b]quinoline 6 as a
result of dehydrative chlorination. The amination of 6 via
an SNAr reaction smoothly proceeded by heating with
amines in THF to give various 11-aminoindolo[2,3-
b]quinolines 7–9 as depicted in Scheme 1.
We introduced an ester group to the indole motif at the
C-9 position, and varied the C-11 position with different
amines (8a–8g). The results of the anti-proliferative eval-
uation against the human leukemia cell line (MV4-11) are
summarized in Table 1. We found that in some cases, their
activities were improved compared to those compounds
without substituents at C-9. For example, 8a, 8b, 8d, and
8e were shown to be more active compared to compounds
10a, 10b, 10d, and 10e which lack a substitutent at the C-2
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Med Chem Res (2013) 22:4492–4504
4495
CO2Me
CO2H
Table 2 The anti-proliferative activity of agents 9 against
human leukemia cell line MV4-11
R2
R2
R1
R1
IC50 (µM)
Compound 9
R1
R2
R3
Yield%
95
N
N
N
N
MV4-11
Me
Me
HN
NH2
NH2
NH2
NH2
NH2
–CO2Me
–CO2Me
–CO2Me
–Br
0.133 0.057
9a
9b
9c
9d
9e
–CO2Me
–Br
8
11
HN
HN
HN
HN
83
0.050 0.016
0.056 0.035
0.118 0.075
0.076 0.034
Scheme 2 Preparation of the 9-caboxylic acid substituted indolo[2,3-
b]quinolines 11. Reagents and conditions: MeOH/NaOH(aq)
–Cl
88
–CO2Me
–Cl
96
–Br
91
Table 3 The anti-proliferative activity of agents 11 against human
leukemia cell line MV4-11
IC50 (µM)
Compound
11
R1
H
R2
R3
Yield%
82
MV4-11
>25
or C-9 position. Their IC50 values ranged from 0.044
HN
NH2
–CO2H
–CO2H
11a
to 0.811 lM, and the best result was achieved with a
3-aminopropylamino group substituted at C-11 (8b, IC50
=
Me
11b
H
79
9.28 1.74
N
HN
Me
0.044 lM). Compared with the agent 7, which contained an
ester group at the C-2 position on the quinoline subunit, the
agent 8b was twice more efficient.
Me
11c
11d
H
H
–CO2H
–CO2H
85
76
>25
>25
HN
N
Me
Furthermore, we have synthesized and evaluated agents
9a–9e, bearing the same aminopropylamino group R2 while
varying the ester group at R1 or R2 for the SAR study, and a
synergistic effect were detected (Table 2). In general, an
ester substituent at the C-9 position can efficiently favor the
anticancer activity of the agent while the ester substituent at
the C-2 position is not as efficient. Comparing compounds
9b and 9d for instance, the compound 9b was twice more
active than 9d, the same trend as observed for the agents 7
and 8b. Compared with 8b, the introduction of a substituent
at the C-2 position, compounds 9a, 9b, and 9c for instance,
has made the compound less active.
HN
OH
(human lung cancer cell line A549, human colon cancer
cell line HCT116) and a kind of normal cell line (normal
mice fibroblast cell line BALB/3T3) were chosen for the
tests. Most of the tested compounds were cytotoxic
against the cancer cell lines, except for the agent 8c with
an IC50 value of more than 5 lM. In general, these tested
compounds were selectively cytotoxic against the human
colon cancer HCT116 cell line, and had a lower cyto-
toxicity against the A549 or BALB/3T3 cell lines. Their
IC50 values against the HCT 116 cell line were generally
4–5 times lower than those against the A549 or BALB/
3T3 cell lines. The agents 7, 8a, 8b, 8g, and the 9
series were shown to be more efficient than the wide
spectrum anticancer medicine, doxorubicin, against the
HCT116 cells. In addition, the agent 9c revealed
a better activity against the A549 cells when compared to
doxorubicin.
The solubility of an agent in an aqueous medium can
significantly influence its further application, despite its
high anticancer activity. In order to improve the water
solubility of the agents, we tried to hydrolyze the 9-ester
substituted neocryptolepines into a 9-carboxylic acid
analog by treating the methyl 11-amino-5H-indolo[2,3-
b]quinoline-9-carboxylate 8 with NaOH(aq) in MeOH. The
mixture was refluxed overnight to afford the 9-carboxylic
acid substituted indolo[2,3-b]quinoline 11 in good yield, as
shown in Scheme 2. The evaluation results of their anti-
proliferative activity are summarized in Table 3.
Unfortunately, the anti-proliferative activity of the
hydrolyzed derivatives 11 turned out that though the sol-
ubility of the agents in an aqueous medium was improved,
their anti-proliferative activities dramatically decreased
compared to their precursors 8a–8d.
Spectroscopic characterization of neocryptolepine
derivative 8b interacting with salmon fish
sperm DNA
Furthermore, the indoloquinoline derivative compounds
bearing an ester substituent (7–9) were also evaluated
against other cell lines to evaluate the selective anticancer
properties (Table 4). Two kinds of cancer cell lines
The DNA binding studies of compounds 8b was performed
using UV–vis absorption spectroscopy with salmon fish
sperm DNA in phosphate buffer of pH 7.0 at 20 °C. The
123

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Med Chem Res (2013) 22:4492–4504
Table 4 The test of compounds
8 and 9 as anti-proliferative
agents against A549, HCT116,
and BALB/3T3 cell lines
IC50 (µM)
Compound 8
R1
R2
R3
A549a
HCT116a
BALB/3T3a
Doxorubicin
HCl
0.329 0.097
0.390 0.098
1.078 0.033
8a
–CO2Me
0.587 0.325
0.173 0.047
0.873 0.058
HN
HN
OH
H
H
NH2
–CO2Me
–CO2Me
0.820 0.456
5.09 2.96
0.176 0055
1.31 0.155
0.933 0.047
>25
8b
8c
O
N
H
H
CH3
8d
–CO2Me
3.39 0.800
0.672 0.400
8.21 0.453
N
N
NH2
8e
8f
–CO2Me
–CO2Me
0.712 0.112
0.797 0.125
0.308 0.112
0.444 0.321
0.936 0.024
1.09 0.319
HN
HN
H
H
Me
N
Me
Me
8g
–CO2Me
0.664 0.154
0.151 0.026
0.876 0.023
HN
N
H
Me
–CO2Me
–Br
HN
HN
HN
HN
HN
HN
NH2
–CO2Me
–CO2Me
–CO2Me
–Br
0.864 0.221
0.302 0.138
0.194 0.063
0.461 0.123
0.464 0.111
0.649 0.080
0.295 0.112
0.164 0.070
0.116 0.078
0.163 0.073
0.284 0.142
0.130 0.014
8.40 0.286
0.773 0.023
0.833 0.030
0.743 0.016
0.834 0.067
0.768 0.155
9a
9b
9c
9d
9e
7
NH2
NH2
NH2
NH2
NH2
–Cl
–CO2Me
–Cl
–Br
a
A549 human lung cancer cell
line, HCT116 human colon
cancer, BALB/3T3 normal mice
fibroblast cell line
–CO2Me
H
hypochromic effect was observed in the absorption spectra
while the DNA solution was gradually added to the solution
of the compound 8b. The results in Fig. 2 showed that the
absorption band at 298 nm for the 8b decreased while
increasing the DNA concentration. It illustrated that there is
a strong interaction between 8b and DNA. Then the binding
constant of 8b-DNA was calculated as 2.77 9 105 L mol-1
according to double-reciprocal equation.
Fig. 2 UV–Vis absorption
12
1.4
spectra of compound 8b and
1.2
10
8b-DNA at 20 °C. C8b = 50
y = 2E-06x + 0.5534
1
0.8
0.6
0.4
0.2
0
lmol/L, CDNA = 0.0, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0 lmol/L for curve 1–11 in
pH 7.0 phosphate buffer
solution; (b) the plot of 1/DA vis
1/[DNA] for 8b-DNA
8
R² = 0.9959
6
4
2
0
270
290
310
330
350
370
390
0
2000000
4000000
6000000
wavelength/nm
1/[DNA](mol/L)
123

Med Chem Res (2013) 22:4492–4504
4497
Conclusion
(dd, J = 8.6, 1.7 Hz, 1H), 7.57 (dd, J = 8.6, 0.5 Hz, 1H),
3.87 (s, 3H), 3.84 (s, 3H), 3.34 (s, 2H). 13C NMR
(150 MHz, DMSO-d6) dC (ppm) 167.0, 164.4, 139.0,
134.3, 125.2, 123.3, 122.7, 112.5, 107.4, 51.9, 50.9.
To summarize, we have synthesized a series of C-2 and/or
C-9 ester substituted 11-amino-5-methyl-5H-indolo[2,3-
b]quinolines and evaluated their antiproliferative activity
for an SAR study. The results indicated that the activities
of the agents could be improved by introducing an ester
substituent at the C-9 position. Among them, the methyl
11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quin-
oline-9-carboxylate (8b) was the most potent with an IC50
value of 0.044 lM against the human leukemia MV4-11
cell line. While compound 9a showed 28 times more
selective cytotoxic activity against the human colon cancer
cell line than normal mice fibroblast cell line, BALB/3T3.
Methyl 5-bromo-1H-indole-3-carboxylate 2c
White solids; 1H NMR (400 MHz, CDCl3) dH (ppm) 8.68 (s,
1H), 8.33 (d, J = 1.9 Hz, 1H), 7.92 (d, J = 3.0 Hz, 1H), 7.37
(dd, J = 8.6, 1.9 Hz, 1H), 7.29 (dd, J = 8.6, 0.4 Hz, 1H),
3.93 (s, 3H). 13C NMR (100 MHz, CDCl3) dC (ppm) 165.1,
134.6, 131.8, 127.4, 126.3, 124.2, 115.7, 112.9, 108.6, 51.3.
Methyl 2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-
1H-indole-3-carboxylate 4a
Experimental
White solids, Mp: 217–219 °C; 1H NMR (600 MHz, DMSO-
d6): dH (ppm) 12.25 (br s, 1 H), 8.00 (d, J = 7.92 Hz, 1 H),
7.81 (d, J = 8.80 Hz, 2 H), 7.39 (d, J = 7.63 Hz, 1 H),
7.29–7.15 (m, 2 H), 6.73 (d, J = 8.80 Hz, 2 H), 3.78 (s, 3 H),
3.66 (s, 3 H), 3.37 (s, 3 H); 13C NMR (150 MHz, DMSO-d6)
dC (ppm) 166.1, 163.5, 151.7, 145.3, 132.6, 130.7, 125.8,
122.7,121.5,121.0,119.0,112.9,111.8,98.4, 51.6, 50.6, 39.1.
Chemistry
General
The commercially obtained reagents were directly used
1
without further purification. The H NMR and 13C NMR
spectra were measured on the Varian INOVA-600 or Varian
INOVA-400 spectrometer. High resolution mass spectra
were obtained on a Bruker micrOTOF II-SKA spectrometer.
Melting points were determined on a J-Science RFS-10 hot
stage microscope. The scaffolds 7–8 and were prepared by
the method we previously mentioned (Wang et al., 2013).
Dimethyl 2-(methyl(phenyl)amino)-1H-indole-3,5-
dicarboxylate 4b
White solids, Mp: 168–169 °C; 1H NMR (400 MHz, CDCl3)dH
(ppm) 9.09 (s, 1H), 8.80–8.75 (m, 1H), 7.85(dd, J = 8.5, 1.7 Hz,
1H), 7.25–7.19 (m, 3H), 6.96–6.85 (m, 3H), 3.89 (s, 3H), 3.79 (s,
3H), 3.44 (s, 3H). 13C NMR (100 MHz, CDCl3) dC (ppm) 168.2,
164.3, 149.3, 146.5, 134.9, 129.3, 126.3, 124.0, 123.5(2C), 121.3,
117.1, 110.3, 97.6, 51.9, 51.0, 40.2. ESI-HRMS: m/z Calcd. for
C19H17N2O4 [M–H]- 337.1188. Found 337.1166.
General procedure for the synthesis of Dimethyl
1H-indole-3,5-dicarboxylate 2b
Pyridine (0.30 mL, 3.54 mmol) was added to a suspension
of methyl 1H-indole-5-carboxylate (0.48 g, 2.72 mmol) in
anhydrous THF (6 mL) at 0 °C. A solution of trichloro-
acetyl chloride (0.40 mL, 3.54 mmol) in THF (6 mL) was
added dropwise via an addition funnel over 1 h. The
reaction mixture was then allowed to warm to room tem-
perature to stir over night. The reaction mixture was
quenched in 1 M HCl, dried over Na2SO4, and concen-
trated under vacuum. The resulting solid was then dis-
solved in MeOH (54 mL), and KOH(s) was added. The
reaction mixture was heated to reflux for 5 h, then stirred at
ambient temperature for 1 h, followed by concentration
under vacuum. The solid was purified by chromatography
(SiO2, 25 % EtOAc/Hexane) in 75 % yield.
Dimethyl 2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-
1H-indole-3,5-dicarboxylate 4c
White solids, Mp: 234–236 °C; 1H NMR (400 MHz,
CDCl3) dH (ppm) 9.58 (s, 1H), 8.84 (d, J = 1.3 Hz, 1H),
7.96 (dd, J = 8.5, 1.6 Hz, 1H), 7.80–7.73 (m, 2H), 7.38 (d,
J = 8.5 Hz, 1H), 6.70–6.63 (m, 2H), 3.93 (s, 3H), 3.82 (d,
J = 3.5 Hz, 6H), 3.42 (s, 3H). 13C NMR (100 MHz,
CDCl3) dC (ppm) 168.0, 167.3, 163.9, 150.9, 146.7, 135.0,
131.1, 125.7, 124.7, 124.4, 124.0, 120.6, 113.5, 110.9,
100.8, 52.0, 51.8, 51.2, 39.7. ESI-HRMS: m/z Calcd. for
C21H20N2O6 [M?Na]? 419.1219. Found 419.1217.
Dimethyl 2-((4-chlorophenyl)(methyl)amino)-1H-indole-
Dimethyl 1H-indole-3,5-dicarboxylate 2b
3,5-dicarboxylate 4d
1
White solids; H NMR (400 MHz, DMSO-d6) dH (ppm)
12.28 (s, 1H), 8.69 (d, J = 1.5 Hz, 1H), 8.23 (s, 1H), 7.83
White solids, Mp: 188–189 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 8.79 (s, 1H), 8.70 (s, 1H), 7.92 (dd,
123

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Med Chem Res (2013) 22:4492–4504
J = 8.5, 1.6 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.21–7.16
(m, 2H), 6.82–6.77 (m, 2H), 3.93 (s, 4H), 3.84 (s, 3H), 3.43
(s, 4H). 13C NMR (100 MHz, CDCl3) dC (ppm) 168.01,
164.1, 148.4, 145.4, 134.8, 129.1, 126.1, 125.9, 124.3,
123.8(2C), 117.5, 110.4, 98.8, 52.08, 51.1, 40.1. ESI-
HRMS: m/z Calcd. for C19H18ClN2O4 [M?H]? 373.0960.
Found 373.0965.
(s, 3H); 13C NMR (150 MHz, DMSO-d6): dC (ppm) 171.0,
165.9, 146.9, 142.0, 134.7, 130.9, 127.8, 124.2, 123.7,
123.2, 122.5, 121.5, 120.3, 115.8, 111.0, 102.9, 52.1, 33.7;
Methyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-
b]quinoline-9-carboxylate 5b
Pale gray solids, Mp:[300 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 12.45 (s, 1H), 8.86–8.81 (m, 1H),
8.40 (d, J = 7.8 Hz, 1H), 7.94–7.86 (m, 1H), 7.78 (q,
J = 9.1 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.42 (t,
J = 7.1 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) dC (ppm) 171.6, 166.9, 147.8,
139.2, 137.9, 131.5, 125.8, 124.7, 124.2, 123.8, 122.5,
122.0, 121.6, 115.5, 110.7, 102.1, 51.9, 33.4. ESI-HRMS:
m/z Calcd. for C18H13N2O3 [M-H]- 305.0932. Found
305.0867.
Dimethyl 2-((4-bromophenyl)(methyl)amino)-1H-indole-
3,5-dicarboxylate 4e
White solids, Mp: 193–194 °C; 1H NMR (400 MHz,
CDCl3) dH (ppm) 8.85 (s, 1H), 8.79 (s, 1H), 7.91 (dd,
J = 8.5, 1.6 Hz, 1H), 7.34–7.26 (m, 3H), 6.75–6.67 (m,
2H), 3.92 (s, 3H), 3.83 (s, 3H), 3.41 (s, 3H). 13C NMR
(100 MHz, CDCl3) dC (ppm) 168.0, 164.0, 148.1, 145.9,
134.7, 132.1, 126.1, 124.4, 123.9(2C), 117.7, 113.2, 110.4,
99.1, 52.0, 51.1, 40.0. ESI-HRMS: m/z Calcd. for
C19H18BrN2O4 [M?H]? 417.0450. Found 417.0452.
Dimethyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-
b]quinoline-2,9-dicarboxylate 5c
Methyl 5-bromo-2-((4-
(methoxycarbonyl)phenyl)(methyl)amino)-1H-indole-3-
carboxylate 4f
Pale gray solids, Mp:[300 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 12.53 (s, 1H), 8.91 (d, J = 2.1 Hz,
1H), 8.78 (d, J = 1.3 Hz, 1H), 8.17 (dd, J = 8.9, 2.2 Hz,
1H), 7.89 (dd, J = 8.4, 1.7 Hz, 1H), 7.82 (d, J = 9.0 Hz,
1H), 7.51 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H), 3.91 (d,
J = 4.7 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) dC
(ppm) 171.1, 166.9, 165.9, 147.8, 142.0, 137.8, 131.2
127.7, 124.6, 124.1, 123.4, 122.8(2C), 121.8, 116.1, 111.0,
102.7, 52.2, 52.0, 33.8. ESI-HRMS: m/z Calcd. for
C20H17N2O5 [M?H]? 365.1137. Found 365.1140.
White solids, Mp: 217–218 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 9.08 (s, 1H), 8.29 (d, J = 1.9 Hz, 1H),
7.81–7.76 (m, 2H), 7.37 (dd, J = 8.5, 2.0 Hz, 1H), 7.22 (d,
J = 8.6 Hz, 1H), 6.69–6.64 (m, 2H), 3.84 (s, 3H), 3.81 (s,
3H), 3.42 (s, 3H). 13C NMR (150 MHz, CDCl3) dC (ppm)
167.3, 163.8, 151.0, 146.1, 131.1, 130.7, 127.8, 126.4,
124.4, 120.6, 115.7, 113.5, 112.5, 99.7, 51.9, 51.2, 39.7.
ESI-HRMS: m/z Calcd. for C19H18BrN2O4 [M?H]?
417.0450. Found 417.0457.
Methyl 2-chloro-5-methyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-9-carboxylate 5d
Methyl 5-bromo-2-((4-chlorophenyl)(methyl)amino)-1H-
indole-3-carboxylate 4g
Pale gray solids, Mp: [300 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 12.54 (s, 1H), 8.81 (s, 1H), 8.29 (s,
1H), 7.92 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H),
7.80 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 3.98 (s,
3H), 3.90 (s, 3H).13C NMR (150 MHz, DMSO-d6) dC
(ppm) 170.3, 166.8, 147.9, 138.0(2C), 131.2, 126.9, 125.9,
124.6(2C), 123.6, 122.7, 121.8, 118.1, 111.0, 102.5, 51.9,
33.8. ESI-HRMS: m/z Calcd. for C18H14ClN2O3 [M?H]?
341.0693. Found 341.0679.
White solids, Mp: 193–194 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 8.36 (s, 1H), 8.24 (d, J = 1.9 Hz, 1H),
7.31 (dd, J = 8.5, 1.9 Hz, 1H), 7.23–7.16 (m, 2H), 7.11 (d,
J = 8.5 Hz, 1H), 6.79–6.75 (m, 2H), 3.82 (s, 3H), 3.41 (s,
3H). 13C NMR (150 MHz, CDCl3) dC (ppm) 164.0, 147.9,
145.5, 130.4, 129.2, 128.2, 125.9(2C), 124.1, 117.5, 115.5,
112.0, 97.9, 51.1, 40.1. ESI-HRMS: m/z Calcd. for
C17H15BrClN2O2 [M?H]? 393.0005. Found 393.0006.
Methyl 2-bromo-5-methyl-11-oxo-6,11-dihydro-5H-
Methyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-
indolo[2,3-b]quinoline-9-carboxylate 5e
b]quinoline-2-carboxylate 5a
Pale gray solids, Mp: [300 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 12.57 (s, 1H), 8.82 (d, J = 1.3 Hz,
1H), 8.44 (d, J = 2.4 Hz, 1H), 7.96–7.87 (m, 2H), 7.81 (d,
J = 9.1 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H),
3.90 (s, 3H). 13C NMR (150 MHz, DMSO-d6) dC (ppm)
Pale gray solids, Mp: [300 °C; 1H NMR (600 MHz,
DMSO-d6): dH (ppm) 12.21 (s, 1H), 8.96 (d, J = 2.05 Hz,
1H), 8.28–8.12 (m, 2H), 7.84 (d, J = 8.80 Hz, 1H), 7.48
(d, J = 7.63 Hz, 1H), 7.36–7.17 (m, 2H), 3.98 (s, 3H), 3.91
123

Med Chem Res (2013) 22:4492–4504
4499
Dimethyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-
170.2, 166.9, 147.9, 138.3, 138.1, 133.9, 127.8, 126.3,
124.6, 123.6, 122.7, 121.8, 118.3, 114.8, 111.0, 102.5,
51.9, 33.7. ESI-HRMS: m/z Calcd. for C18H13BrN2O3
[M?Na]? 407.0007. Found 407.0009.
2,9-dicarboxylate 6c
Orange solids, Mp: 278–280 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 9.14 (d, J = 1.8 Hz, 1H), 9.03 (d,
J = 1.5 Hz, 1H), 8.51 (dd, J = 8.9, 1.8 Hz, 1H), 8.24 (dd,
J = 8.4, 1.6 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.79 (d,
J = 8.4 Hz, 1H), 4.55 (s, 3H), 4.06 (s, 3H), 3.99 (s, 3H).
13C NMR (150 MHz, CDCl3) dC (ppm) 167.2, 165.7,
156.4, 155.7, 139.1, 138.0, 132.2, 131.7, 128.6, 125.8,
125.1, 124.4, 123.0, 122.8, 119.2, 117.1, 115.0, 52.7, 52.1,
34.4. ESI-HRMS: m/z Calcd. for C20H16ClN2O4 [M?H]?
383.0799. Found 383.0799.
Methyl 9-bromo-5-methyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-2-carboxylate 5f
Pale gray solids, Mp: [300 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 12.34 (s, 1H), 8.88 (d, J = 2.1 Hz,
1H), 8.22 (s, 1H), 8.15 (dd, J = 8.8, 2.1 Hz, 1H), 7.79 (d,
J = 9.0 Hz, 1H), 7.38 (d, J = 1.7 Hz, 2H), 3.92 (s, 3H),
3.90 (s, 3H). 13C NMR (150 MHz, DMSO-d6) dC (ppm)
171.1, 165.8, 147.2, 142.0, 133.6, 131.2, 127.7, 125.5(2C),
123.9, 122.7, 122.2, 115.9, 113.7, 113.0, 102.0, 52.2, 33.7.
ESI-HRMS: m/z Calcd. for C18H13BrN2O3 [M?Na]?
407.0007. Found 407.0007.
Methyl 2,11-dichloro-5-methyl-5H-indolo[2,3-b]quinoline-
9-carboxylate 6d
Orange solids, Mp: 283–284 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 8.90 (d, J = 1.2 Hz, 1H), 8.33 (d,
J = 2.3 Hz, 1H), 8.17 (dd, J = 8.4, 1.7 Hz, 1H), 7.73 (dd,
J = 9.1, 2.3 Hz, 1H), 7.63 (dd, J = 21.2, 8.7 Hz, 2H), 4.33
(s, 3H), 3.97 (s, 3H). 13C NMR (150 MHz, CDCl3) dC (ppm)
167.3, 157.6, 155.9, 135.9, 135.1, 131.8, 131.6, 129.2, 125.7,
125.3, 124.7, 122.8, 122.3, 120.4, 117.0, 116.2, 52.1, 33.9.
ESI-HRMS: m/z Calcd. for C18H13Cl2N2O2 [M?H]?
359.0354. Found 359.0357.
9-bromo-2-chloro-5-methyl-5H-indolo[2,3-b]quinolin-
11(6H)-one 5g
Pale gray solids, Mp: [300 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 12.27 (s, 1H), 8.22 (dd, J = 5.2,
1.8 Hz, 2H), 7.77 (d, J = 9.1 Hz, 1H), 7.73 (dd, J = 9.0,
2.6 Hz, 1H), 7.44–7.30 (m, 2H), 3.91 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) dC (ppm) 170.2, 147.3, 137.8,
133.7, 131.0, 126.7, 125.6(2C), 125.4, 124.6, 122.1, 117.8,
113.5, 112.9, 101.7, 33.6. ESI-HRMS: m/z Calcd. for
C16H10BrClN2O [M?Na]? 382.9563. Found 382.9565.
Methyl 2-bromo-11-chloro-5-methyl-5H-indolo[2,3-
b]quinoline-9-carboxylate 6e
Methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-2-
Orange solids, Mp: 289–290 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 9.06 (d, J = 1.5 Hz, 1H), 8.60 (d,
J = 2.2 Hz, 1H), 8.27 (dd, J = 8.4, 1.7 Hz, 1H), 7.96 (dd,
J = 9.1, 2.2 Hz, 1H), 7.74 (dd, J = 20.0, 8.7 Hz, 2H), 4.46
(s, 3H), 3.99 (s, 3H). 13C NMR (150 MHz, CDCl3) dC (ppm)
167.5, 158.5, 156.5, 135.7, 134.4, 131.7, 130.9, 128.8, 128.5,
125.9, 125.2, 123.3, 122.2, 120.8, 117.3, 116.3, 52.1, 33.6.
ESI-HRMS: m/z Calcd. for C18H13BrClN2O2 [M?H]?
402.9849. Found 402.9848.
carboxylate 6a
Orange solids, Mp: 281–283 °C; 1H NMR (600 MHz,
CDCl3): dH (ppm) 8.95 (s, 1H), 8.29 (d, J = 8.22 Hz, 2H),
7.74–7.57 (m, 2H), 7.52 (t, J = 7.48 Hz, 1H), 7.22 (t,
J = 7.48 Hz, 1H), 4.24 (s, 3H), 4.00 (s, 3H); 13C NMR
(150 MHz, CDCl3): dC (ppm) 166.0, 155.1, 154.5, 139.2,
135.7, 131.3, 130.1, 128.2, 125.4, 123.9, 123.8,123.5,
120.9, 118.5, 117.8, 114.3, 52.4, 33.4.
Methyl 9-bromo-11-chloro-5-methyl-5H-indolo[2,3-
Methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-9-
b]quinoline-2-carboxylate 6f
carboxylate 6b
Orange solids, Mp:[300 °C; 1H NMR (600 MHz, CDCl3)
dH (ppm) 9.14 (d, J = 1.8 Hz, 1H), 8.53 (d, J = 1.9 Hz,
1H), 8.48 (dd, J = 8.9, 1.9 Hz, 1H), 7.84 (d, J = 9.0 Hz,
1H), 7.70–7.60 (m, 2H), 4.43 (s, 3H), 4.04 (d, J = 8.0 Hz,
3H). 13C NMR (150 MHz, CDCl3) dC (ppm) 166.0, 155.4,
153.4, 139.6, 137.9, 137.2, 132.8, 132.0, 128.7, 126.5, 124.7,
124.3, 119.4, 118.7, 114.7, 113.6, 52.6, 33.6. ESI-HRMS:
m/z Calcd. for C18H13BrClN2O2 [M?H]? 402.9849. Found
402.9850.
Orangesolids, Mp:248–249 °C;1HNMR(400 MHz,CDCl3)
dH (ppm) 9.01 (s, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.20 (d,
J = 8.4 Hz, 1H), 7.88 (t, J = 7.7 Hz, 1H), 7.81 (d,
J = 8.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.60 (t,
J = 7.6 Hz, 1H), 4.43 (d, J = 7.3 Hz, 3H), 3.98 (s, 3H). 13
C
NMR (150 MHz, CDCl3) dC (ppm) 167.4, 157.6, 156.1,
137.1, 136.5, 131.5, 130.9, 126.0, 125.4, 123.4, 122.9(2C),
121.5, 119.2, 116.7, 114.4, 51.9, 33.4. ESI-HRMS: m/z Calcd.
for C18H14ClN2O2 [M?H]? 325.0744. Found 325.0797.
123

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Med Chem Res (2013) 22:4492–4504
9-Bromo-2,11-dichloro-5-methyl-5H-indolo[2,3-
116.0, 115.6(d), 103.1, 58.4, 51.6, 45.8, 40.0(overlap with
DMSO-d6 peaks), 33.6, 32.5. ESI-HRMS: m/z Calcd. for
C21H20N3O3[M-H]- 362.1492. Found 362.1483.
b]quinoline 6g
Orange solids, Mp: 288–290 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) 8.45 (d, J = 1.9 Hz, 1H), 8.37 (d,
J = 2.3 Hz, 1H), 7.76 (dd, J = 9.1, 2.4 Hz, 1H), 7.66 (d,
J = 9.1 Hz, 1H), 7.62 (dd, J = 8.4, 2.0 Hz, 1H), 7.54 (d,
J = 8.4 Hz, 1H), 4.30 (s, 3H). 13C NMR (150 MHz,
CDCl3) dC (ppm) 155.0, 153.6, 135.6, 135.5, 132.8, 131.8,
128.6, 126.5, 125.4, 125.0, 124.8, 120.08, 119.1, 115.9,
113.1, 33.5. ESI-HRMS: m/z Calcd. for C16H10BrCl2N2
[M?H]? 378.9404. Found 378.9406.
Methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-
b]quinoline-9-carboxylate 8b
Yellow solids, Mp: 170–171 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 8.63 (d, J = 1.4 Hz, 1H), 8.52 (d,
J = 7.7 Hz, 1H), 7.93–7.87 (m, 2H), 7.83 (t, J = 7.7 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.4 Hz, 1H), 4.19
(s, 3H), 4.00 (t, J = 6.6 Hz, 2H), 3.86 (s, 3H), 2.71 (t,
J = 6.3 Hz, 2H), 1.84–1.78 (m, 2H); 13C NMR (150 MHz,
DMSO-d6) dC (ppm) 167.4, 158.3, 155.7, 149.1, 137.5,
131.1, 125.6, 124.1(2C), 123.6, 121.2, 118.3, 116.0,
115.5(2C), 102.6, 51.6, 47.2, 33.1, 32.5. ESI-HRMS: m/z
Calcd. for C21H21N4O2[M-H]- 361.1670. Found 361.1639.
General procedure for the synthesis of ester substituted
11-amino-5-methyl-5H-indolo[2,3-b]quinolines 7–9
The methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-
2-carboxylate (6) or methyl 11-chloro-5-methyl-5H-indo-
lo[2,3-b]quinoline-9-carboxylate (7) was heated at reflux
with a large excess of an appropriate amine in THF for 3–4 h.
The reaction was monitored by TLC. Then the mixture was
washed with water and extracted with CH2Cl2. The organic
phase was dried over MgSO4 and concentrated under vac-
uum. The crude product was purified by column chroma-
tography using eluent changed from AcOEt to AcOEt-2 N
ammonia in MeOH (10: 1) to give the final product.
Methyl 5-methyl-11-morpholino-5H-indolo[2,3-
b]quinoline-9-carboxylate 8c
Orange solids, Mp: 213–215 °C; 1H NMR (400 MHz,
CDCl3) dH (ppm) 9.01 (d, J = 1.6 Hz, 1H), 8.50 (d,
J = 8.2 Hz, 1H), 8.20 (dd, J = 8.4, 1.7 Hz, 1H), 7.83 (d,
J = 4.1 Hz, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.57–7.47 (m,
1H), 4.41 (s, 3H), 4.18–4.09 (m, 4H), 3.98 (s, 3H),
3.75–3.66 (m, 4H); 13C NMR (100 MHz, CDCl3) dC
(ppm) 167.8, 158.4, 156.7, 151.3, 138.1, 131.0, 129.7,
126.5, 126.0, 122.4, 122.0, 121.0(2C), 120.7, 116.5, 115.1,
67.6, 51.9, 51.0, 33.7. ESI-HRMS: m/z Calcd. for
C22H22N3O3 [M?H]? 376.1661. Found 376.1662.
Methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-
b]quinoline-2-carboxylate 7
Yellow solids, Mp: 149–150 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 9.12 (d, J = 1.8 Hz, 1 H), 8.25 (dd,
J = 9.0, 1.8 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.90 (d,
J = 9.1 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.29 (t,
J = 7.6 Hz, 1H), 7.09 (t, J = 7.0 Hz, 1H), 4.17 (s, 3H),
3.99 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.67 (t, J = 6.3 Hz,
2H), 1.78–1.74 (m, 2H); 13C NMR (150 MHz, DMSO-d6)
dC (ppm) 165.9, 156.2, 151.9, 148.1, 140.2, 130.2, 126.5,
124.5, 124.3, 122.1, 121.3, 118.5, 116.9, 115.3, 115.1,
103.7, 52.2, 47.4, 39.9, 33.1, 32.5. ESI-HRMS: m/z Calcd.
for C21H21N4O2 [M-H]- 361.1670. Found 375.1666.
Methyl 5-methyl-11-(4-methyl-1,4-diazepan-1-yl)-5H-
indolo[2,3-b]quinoline-9-carboxylate 8d
Orange solids, Mp: 179–180 °C; 1H NMR (400 MHz,
CDCl3) dH (ppm) 8.93 (s, 1H), 8.47 (d, J = 8.2 Hz, 1H),
8.26–8.18 (m, 1H), 7.80 (dd, J = 3.7, 1.7 Hz, 2H), 7.71 (d,
J = 8.4 Hz, 1H), 7.50 (ddd, J = 8.1, 4.2, 1.7 Hz, 1H), 4.37
(d, J = 0.8 Hz, 3H), 3.97 (d, J = 0.9 Hz, 3H), 3.93–3.87
(m, 2H), 3.78 (t, J = 6.1 Hz, 2H), 3.14 (d, J = 4.5 Hz,
2H), 3.05–2.90 (m, 2H), 2.63 (s, 3H), 2.39–2.23 (m, 2H);
13C NMR (100 MHz, CDCl3) dC (ppm) 168.1, 159.5,
157.8, 152.3, 138.5, 130.7, 129.7, 126.8, 125.7, 122.8,
122.0, 121.5, 121.0, 120.8, 116.8, 115.0, 61.3, 57.9, 52.9,
52.2, 51.9, 47.2, 33.3, 29.2. ESI-HRMS: m/z Calcd. for
C24H27N4O2 [M?H]? 403.2134. Found 403.2135.
Methyl 11-(3-hydroxypropylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate 8a
Orange solids, Mp: 223–224 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 8.60–8.50 (m, 2H), 7.94–7.88 (m,
2H), 7.86–7.81 (m, 1H), 7.52 (d, J = 8.4 Hz, 1H),
7.50–7.43 (m, 2H), 4.57 (t, J = 4.7 Hz, 1H), 4.19 (s, 3H),
3.97 (q, J = 6.5 Hz, 2H), 3.87 (s, 3H), 3.47 (dd, J = 10.8,
5.9 Hz, 2H), 1.90 (p, J = 6.4 Hz, 2H); 13C NMR
(150 MHz, DMSO-d6) dC (ppm) 167.4, 158.3, 155.8,
149.2, 137.4, 131.2, 125.7, 124.0(2C), 123.6, 121.3, 118.4,
Methyl 11-(4-aminobutylamino)-5-methyl-5H-indolo[2,3-
b]quinoline-9-carboxylate 8e
Yellow oil; 1H NMR (600 MHz, DMSO-d6) dH (ppm) 8.62
(t, J = 6.1 Hz, 1H), 8.53 (s, 1H), 7.92 (dd, J = 8.3, 7.0, 2H),
123

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4501
Methyl 11-(3-aminopropylamino)-2-chloro-5-methyl-5H-
7.85 (t, J = 7.7 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.49 (t,
J = 7.5 Hz, 1 H), 4.20 (s, 3H), 3.90 (t, J = 7.1 Hz, 2H), 3.87
(s, 3H), 2.67 (t, J = 7.4 Hz, 2H), 1.85–1.76 (m, 2H), 1.48 (d,
J = 6.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) dC
(ppm) 167.4, 158.4, 155.9, 149.0, 137.4, 131.2, 125.8, 124.0
(2C), 123.7, 121.4, 118.4, 116.1, 115.7, 115.5, 103.0, 51.7,
47.3, 38.6, 32.5, 27.6, 24.9. ESI-HRMS: m/z Calcd. for
C22H25N4O2 [M ? H]? 377.1978. Found 377.1978.
indolo[2,3-b]quinoline-9-carboxylate 9b
Yellow solids, Mp: 185–186 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 8.59 (d, J = 2.0 Hz, 1H), 8.54 (s,
1H), 7.89 (dd, J = 8.4, 1.4 Hz, 1H), 7.84 (d, J = 9.2 Hz,
1H), 7.79 (dd, J = 9.1, 2.1 Hz, 1H), 7.50 (d, J = 8.3 Hz,
1H), 4.12 (s, 3H), 3.93 (t, J = 6.7 Hz, 2H), 3.86 (s, 3H),
2.67 (t, J = 6.4 Hz, 2H), 1.86–1.77 (m, 2H). 13C NMR
(150 MHz, DMSO-d6) dC (ppm) 167.4, 158.1, 155.8,
147.9, 136.1, 130.7, 125.8(2C), 123.9, 123.2, 118.6, 117.5,
116.7, 116.2, 104.6, 103.1, 51.8, 46.8, 39.9(overlap with
DMSO-d6 peaks), 33.3, 32.6. ESI-HRMS: m/z Calcd. for
C21H22ClN4O2 [M?H]? 397.1431. Found 397.1432.
Methyl 11-(2-(dimethylamino)ethylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate 8f
Yellow solids, Mp: 172–173 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) = 8.70 (s, 1H), 8.54 (d, J = 8.3 Hz,
1H), 7.97–7.91 (m, 2H), 7.86 (t, J = 7.8 Hz, 1H), 7.55 (d,
J = 8.4 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.26 (t,
J = 4.5 Hz, 1H), 4.21 (s, 3H), 4.00 (dd, J = 10.8, 5.5 Hz,
2H), 3.87 (s, 3H), 2.65 (t, J = 6.1 Hz, 2H), 2.27 (s, 6H);
13C NMR (150 MHz, CDCl3) dC (ppm) = 168.2, 157.4,
155.6, 149.3, 138.3, 130.5, 127.0, 125.3, 123.8, 122.5,
120.6, 119.7, 116.1, 115.6, 114.8, 106.0, 58.2, 51.5, 45.2,
44.7, 32.6. ESI-HRMS: m/z Calcd. for C22H23N4O2 [M-H]-
375.1826. Found 375.1806.
Methyl 11-(3-aminopropylamino)-2-bromo-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate 9c
Yellow solids, Mp: 109–112 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 8.76 (s, 1H), 8.57 (s, 1H), 7.97–7.88
(m, 2H), 7.84 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 8.4 Hz,
1H), 4.16 (s, 3H), 3.95 (t, J = 6.7 Hz, 2H), 3.86 (s, 3H),
2.67 (t, J = 6.4 Hz, 2H), 1.82 (t, J = 6.5 Hz, 2H). 13C
NMR (150 MHz, DMSO-d6) dC (ppm) 167.4, 158.1, 155.8,
147.9, 136.4, 133.4, 126.1, 125.9, 123.9(2C), 118.6, 117.7,
117.3, 116.2, 113.5 103.1, 51.7, 46.6, 39.3, 33.1, 32.6, 32.3.
ESI-HRMS: m/z Calcd. for C21H22BrN4O2 [M?H]?
441.0926. Found 441.0927.
Methyl 11-(3-(dimethylamino)propylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate 8g
Yellow solids, Mp: 105–106 °C; 1H NMR (600 MHz,
CDCl3) dH (ppm) = 8.65 (d, J = 1.4 Hz, 2H), 8.07 (dd,
J = 8.4, 1.5 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.70 (dd,
J = 11.9, 4.4 Hz, 2H), 7.65 (d, J = 8.6 Hz, 1H), 7.35 (t,
J = 7.6 Hz, 1H), 4.23 (d, J = 1.2 Hz, 3H), 4.20–4.10 (m,
2H), 3.94 (s, 3H), 2.72–2.64 (m, 2H), 2.39 (s, 6H),
1.95–1.73 (m, 2H); 13C NMR (150 MHz, CDCl3) dC
(ppm) = 168.4, 158.3, 155.5, 149.0, 137.5, 130.3, 126.2,
124.1, 123.6, 123.4, 120.7, 119.1, 115.9, 115.8, 114.5,
103.0, 59.5, 51.5, 50.1, 45.4, 32.5, 26.3. ESI-HRMS: m/z
Calcd. for C23H25N4O2[M-H]- 389.1983. Found
389.1949.
Methyl 11-(3-aminopropylamino)-9-bromo-5-methyl-5H-
indolo[2,3-b]quinoline-2-carboxylate 9d
Yellow solids, Mp: 162–163 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 9.10 (d, J = 1.6 Hz, 1H), 8.24 (dd,
J = 8.9, 1.7 Hz, 1H), 8.10 (d, J = 1.9 Hz, 1H), 7.88 (d,
J = 9.0 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.39 (dd,
J = 8.4, 1.9 Hz, 1H), 4.14 (s, 3H), 3.97 (t, J = 6.6 Hz, 2H),
3.92 (s, 3H), 2.71 (t, J = 6.2 Hz, 2H), 1.83–1.77 (m, 2H).
13C NMR (150 MHz, DMSO-d6) dC (ppm) 165.8, 156.5,
150.5, 149.0, 140.4, 130.7, 126.7(2C), 126.2, 124.0, 121.5,
118.5, 115.6, 114.9, 110.4, 102.3, 52.2, 47.5, 39.9(overlap
with DMSO-d6 peaks), 32.9, 32.6. ESI-HRMS: m/z Calcd.
for C21H22BrN4O2 [M?H]? 441.0926. Found 441.0927.
Dimethyl 11-(3-aminopropylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-2,9-dicarboxylate 9a
Yellow solids, Mp: 75–76 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 9.17 (s, 1H), 8.63 (s, 1H), 8.28 (d,
J = 8.1 Hz, 1H), 7.97–7.89 (m, 2H), 7.55 (d, J = 8.4 Hz,
1H), 4.20 (s, 3H), 4.02 (t, J = 6.6 Hz, 2H), 3.94 (s, 3H),
3.87 (s, 3H), 2.75 (t, J = 6.3 Hz, 2H), 1.87–1.82 (m, 2H).
13C NMR (150 MHz, DMSO-d6) dC (ppm) 167.4, 165.9,
158.2, 155.4, 148.9, 140.2, 130.7, 126.6, 125.7, 124.1,
123.7, 122.0, 119.0, 116.4, 115.8, 115.2, 102.5, 52.3,
51.6(2C), 47.1, 32.8, 32.5. ESI-HRMS: m/z Calcd. for
C23H25N4O4 [M?H]? 421.1876. Found 421.1877.
N-(3-aminopropyl)-9-bromo-2-chloro-5-methyl-5H-
indolo[2,3-b]quinolin-11-amine 9e
Yellow solids, Mp: 152–154 °C; 1H NMR (600 MHz,
DMSO-d6) dH (ppm) 8.59 (d, J = 1.8 Hz, 1H), 8.03 (d,
J = 1.7 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.82 (dd,
J = 9.1, 2.0 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.39 (dd,
J = 8.4, 1.9 Hz, 1H), 4.12 (s, 3H), 3.91 (t, J = 6.6 Hz,
2H), 2.63 (t, J = 6.3 Hz, 2H), 1.77 (p, J = 6.5 Hz, 2H).
123

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Med Chem Res (2013) 22:4492–4504
13C NMR (150 MHz, DMSO-d6) dC (ppm) 156.6, 150.9,
148.1, 136.3, 130.7, 126.9, 126.1, 125.2, 124.2, 123.3,
118.3, 117.3, 116.5, 109.9, 102.9, 46.9, 39.5(overlap with
DMSO-d6 peaks), 33.3, 32.5. ESI-HRMS: m/z Calcd. for
C19H19BrClN4 [M?H]? 417.0482. Found 417.0480.
2H), 3.15–3.04 (m, 2H), 2.70 (s, 6H), 2.31 (dt, J = 14.0,
7.2 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) dC
(ppm)167.7, 152.4, 149.3, 136.8, 133.2, 127.3, 124.9, 124.5
(2C), 124.2, 120.8, 116.9, 116.7, 112.2, 98.6, 53.7, 45.3,
42.1, 35.6, 25.1. ESI-HRMS: m/z Calcd. for C22H23N4O2
[M-H]- 375.1826. Found 375.1828.
General procedure for the synthesis of 11-amino-5-methyl-
5H-indolo[2,3-b]quinoline-9-carboxylic acid 11
11-(3-Hydroxypropylamino)-5-methyl-5H-indolo[2,3-
b]quinoline-9-carboxylic acid 11d
Methyl 11-amino-5-methyl-5H-indolo[2,3-b]quinoline-9-
carboxylate was dissolved in MeOH and refluxed with
20 % NaOH (aq.) over night. The reaction was monitored
by TLC. Then the mixture was neutralized with 1 N HCl
(aq.) and concentrated to remove the MeOH under vacuum.
The crude product was purified by reverse-phase chroma-
tography using eluent changed from H2O to H2O/CH3CN
(50:1) to give carboxylic acid.
1
White solids, Mp: [300 °C; H NMR (600 MHz, DMSO-
d6) dH (ppm) 8.86–8.72 (m, 2H), 8.59 (s, 1H), 8.10 (d,
J = 8.5, 1H), 8.05 (d, J = 8.3, 1H), 7.99 (t, J = 7.6, 1H),
7.76 (d, J = 8.3, 1H), 7.66 (t, J = 7.5, 1H), 4.24 (s, 3H),
4.04 (dd, J = 11.8, 6.0, 2H), 3.46 (t, J = 5.6, 2H),
2.07–1.82 (m, 2H); 13C NMR (150 MHz, DMSO-d6) dC
(ppm) 167.4, 152.4, 148.7, 140.2, 136.7, 132.9, 127.0,
124.8, 124.4, 124.1, 123.9, 120.6, 116.8, 116.5, 111.9,
98.2, 58.0, 46.1, 35.6, 32.8. ESI-HRMS: m/z Calcd. for
C20H18N3O3 [M–H]- 348.1354. Found 348.1327.
11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-
b]quinoline-9-carboxylic acid 11a
White solids, Mp:[300 °C; 1H NMR (600 MHz, D2O) dH
(ppm) 8.10 (d, J = 8.2 Hz, 1H), 7.90 (t, J = 7.5 Hz, 1H),
7.71 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 9.9, 4.9 Hz, 2H),
7.53 (s, 1H), 7.17 (dd, J = 7.9, 4.8 Hz, 1H), 3.69–3.57
(m,5H), 2.93–2.83 (m, 2H), 2.06–1.91 (m, 2H). 13C NMR
(150 MHz, D2O) dC (ppm) 168.9, 151.6, 146.9, 139.0, 135.8,
133.7, 127.4, 125.1, 123.7, 123.3, 122.4, 118.8, 116.2, 115.4,
111.0, 97.9, 44.8, 36.4, 34.3, 27.9. ESI-HRMS: m/z Calcd.
for C20H19N4O2 [M-H]- 347.1513. Found 347.1505.
Antitumor screening test
Cell lines
Established in vitro, human cell line: MV4-11 (leukemia),
A549 (lung cancer), HCT116 (colon cancer), and normal
mice fibroblast (Balb/3T3) were used. These lines were
obtained from American Type Culture Collection (Rock-
ville, Maryland, USA) and were being maintained at the
Institute of Immunology and Experimental Therapy,
Wroclaw, Poland.
11-(2-(dimethylamino)ethylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylic acid 11b
MV4-11 cells were cultured in the RPMI 1640 supple-
mented with 2 mM L-glutamine, 1.0 mM sodium pyruvate
and 10 % fetal bovine serum (all from Sigma-Aldrich
Chemie GmbH, Steinheim, Germany), HCT 116, and A549
cells were cultured in the RPMI 1640 ? OptiMEM (50:50)
medium (Gibco, Scotland, UK) supplemented with 2 mM
L-glutamine and 5 % fetal bovine serum (all from Sigma-
Aldrich Chemie GmbH, Steinheim, Germany), BALB/3T3
cells were cultured in Dulbecco medium (IIET) supple-
mented with 2 mM L-glutamine and 1.0 mM sodium
pyruvate, 10 % fetal bovine serum (all from Sigma-Aldrich
Chemie GmbH, Steinheim, Germany). All culture medium
was supplemented with 100 units/ml penicillin and 100 lg/ml
streptomycin (both from Polfa, Tarchomin S.A., Poland).
All cell lines were grown at 37 °C with 5 % CO2 humidified
atmosphere.
White solids, Mp: 276–280 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) = 9.03 (d, J = 8.0 Hz, 2H), 8.57 (s,
1H), 8.15 (dd, J = 19.0, 8.6 Hz, 2H), 8.04 (t, J = 7.8 Hz,
1H), 7.82 (d, J = 8.4 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H),
4.37 (dd, J = 11.4, 5.7 Hz, 2H), 4.30 (s, 3H), 3.55 (t,
J = 5.5 Hz, 2H), 2.78 (s, 6H). 13C NMR (150 MHz,
DMSO-d6) dC (ppm) 167.6, 152.5, 149.3, 140.71, 136.7,
133.3, 127.7, 125.2, 124.9, 124.5 (2C), 120.6, 116.9, 112.2,
99.5, 56.0, 43.1, 42.8, 35.7, 34.4. ESI-HRMS: m/z Calcd.
for C21H21N4O2 [M-H]- 361.1670. Found 361.1653.
11-(3-(Dimethylamino)propylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylic acid 11c
Pale Yellow solids, Mp: 280–282 °C; 1H NMR (400 MHz,
DMSO-d6) dH (ppm) 8.97 (s, 1H), 8.89 (d, J = 8.5 Hz,
1H), 8.52 (s, 1H), 8.12 (dd, J = 16.6, 8.6 Hz, 2H),
8.05–7.98 (m, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.70 (t,
J = 7.7 Hz, 1H), 4.26 (s, 3H), 4.01 (dd, J = 12.9, 6.5 Hz,
Antiproliferative assay in vitro
Test solutions of the compounds tested (1 mg/ml) were
prepared by dissolving the substances in 100 ll of DMSO
123

Med Chem Res (2013) 22:4492–4504
4503
completed with 900 ll of tissue culture medium. After-
ward, the tested compounds were diluted in culture med-
ium to reach the final concentrations of 10, 1, 0.1, 0.01, and
0.001 lg/ml.
50 % TCA (trichloroacetic acid, Aldrich-Chemie, Ger-
many) on the top of the culture medium in each well. The
plates were incubated at 4 °C for 1 h and then washed five
times with tap water. The background optical density was
measured in the wells filled with culture medium, without
the cells. The cellular material fixed with TCA was stained
with 0.4 % sulforhodamine B (SRB, Sigma, Germany)
dissolved in 1 % acetic acid (POCh, Gliwice, Poland) for
30 min. Unbound dye was removed by rinsing (4x) with
1 % acetic acid. The protein-bound dye was extracted with
10 mM unbuffered Tris base (Sigma, Germany) for deter-
mination of optical density (at 540 nm) in a computer-
interfaced, 96-well microtiter plate reader Multiskan RC
photometer (Labsystems, Helsinki, Finland).
Twenty-four hours before the addition of the tested
compounds, the cells were plated in 96-well plates (Sar-
stedt, Germany) at a density of 1 9 104 cells per well. The
assay was performed after 72 h of exposure to varying
concentrations of the tested agents. The in vitro cytotoxic
effect of all agents was examined using the MTT (MV4-
11) or SRB (A549, HCT116 and Balb/3T3) assay.
The results were calculated as an IC50 (inhibitory
concentration 50)—the dose of tested agent which inhibits
proliferation of 50 % of the cancer cell population. IC
values were calculated for each experiment separately and
mean values SD are presented in the Table 1, 2, 3 and 4.
Each compound in each concentration was tested in trip-
licate in a single experiment, which was repeated 3–5
times.
Acknowledgments We gratefully acknowledge supports by Oka-
yama University and the Advanced Science Research Center for
NMR and EA. We thank MEXT for the scholarship to L.W. We are
thankful to Prof. S. Nakashima and Prof. X.-Q.Yu, Sichuan Univer-
sity, for HRMS analyses, and to Prof. J. Futami for UV measure-
ments. Support by Adaptable and Seamless Technology Transfer
Program of JST and generous gift of various indoles from Air Water
Inc. are highly appreciated.
MTT assay
This technique was applied for the cytotoxicity screening
against leukemia cells growing in suspension culture. An
assay was performed after 72 h exposure to varying con-
centrations (from 0.001 to 10 lg/ml) of the tested agents.
For the last 3–4 h of incubation 20 ll of MTT solution
were added to each well (MTT: 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide; stock solution:
5 mg/ml, Sigma-Aldrich, Germany). The mitochondria of
viable cells reduce the pale yellow MTT to a navy blue
formazan: the more viable cells are present in well, the
more MTT will be reduced to formazan. When incubation
time was completed, 80 ll of the lysing mixture were
added to each well (lysing mixture: 225 ml dimethyl-
formamide, POCh, Gliwice, Poland, 67.5 g sodium dode-
cyl sulfate, Sigma-Aldrich, Germany, and 275 ml of
distilled water). After 24 h, when formazan crystals had
been dissolved, the optical densities of the samples were
read on an Multiskan RC photometer (Labsystems, Hel-
sinki, Finland) at 570 nm wavelength. Each compound in
given concentration was tested in triplicates in each
experiment, which was repeated 3–5 times.
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